In this study, we investigated the interaction between Lactobacillus pentosus HC-2 and Vibrio parahaemolyticus E1 when cocultured in Litopenaeus vannamei in vivo and in vitro. Fluorescence microscopy was used to observe the distribution of these two strains, which were labeled with DAPI and CFDA-SE, respectively, in the gut of the shrimp. Fluorescent imaging revealed that L. pentosus HC-2 competitively excluded V. parahaemolyticus E1 in the intestine of L. vannamei when the shrimp diet contained both these strains. The growth of both bacteria and the expression of quorum-sensing-and virulence-related genes of L. pentosus HC-2 or V. parahaemolyticus E1 were monitored when they were cultured alone or together in vitro. In contrast to L. pentosus HC-2, the population density of V. parahaemolyticus E1 decreased significantly after coculture for 8 h. A transcriptional analysis showed that the expression of the luxS gene in L. pentosus HC-2 increased after 8 h when it was cocultured with V. parahaemolyticus E1, suggesting that LuxS is involved in the competitive exclusion of V. parahaemolyticus E1 by L. pentosus HC-2. Interestingly, the aphA, opaR, and tlh genes of V. parahaemolyticus E1 were significantly upregulated in response to heat-killed L. pentosus HC-2 cells. This suggests that the intracellular components of L. pentosus HC-2 induce the expression of toxicity-related factors in V. parahaemolyticus E1 for a short time in coculture.